Microalgal oils, depending on their degree of unsaturation, can be utilized as either nutritional supplements or fuels; thus, a feedstock with genetically designed and tunable degree of unsaturation is desirable to maximize process efficiency and product versatility. Systematic profiling of ex vivo (in yeast), in vitro, and in vivo activities of type-2 diacylglycerol acyltransferases in Nannochloropsis oceanica (NoDGAT2s or NoDGTTs), via reverse genetics, revealed that NoDGAT2A prefers saturated fatty acids (SFAs), NoDGAT2D prefers monounsaturated fatty acids (MUFAs), and NoDGAT2C exhibits the strongest activity toward polyunsaturated fatty acids (PUFAs). As NoDGAT2A, 2C, and 2D originated from the green alga, red alga, and eukaryotic host ancestral participants of secondary endosymbiosis, respectively, a mechanistic model of oleaginousness was unveiled, in which the indigenous and adopted NoDGAT2s formulated functional complementarity and specific transcript abundance ratio that underlie a rigid SFA:MUFA:PUFA hierarchy in triacylglycerol (TAG). By rationally modulating the ratio of NoDGAT2A:2C:2D transcripts, a bank of N. oceanica strains optimized for nutritional supplement or fuel production with a wide range of degree of unsaturation were created, in which proportion of SFAs, MUFAs, and PUFAs in TAG varied by 1.3-, 3.7-, and 11.2-fold, respectively. This established a novel strategy to simultaneously improve productivity and quality of oils from industrial microalgae.

Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL, encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indicating involvement of FliL in the surface sensing pathway. In the present study, we compared the fliL transcriptome with that of wild-type swarmer cells and showed that nearly all genes associated with motility (flagellar class II and III genes) and chemotaxis are repressed. In contrast, spontaneous motile revertants of fliL cells that regained motility yet produced differentiated swarmer cells under noninducing conditions transcribed flagellar class II promoters at consistent levels. Expression of umoA (a known regulator of swarmer cells), flgF, and flgI increased significantly in both swarmer and pseudoswarmer cells, as did genes in a degenerate prophage region situated immediately adjacent to the Rcs phosphorelay system. Unlike swarmer cells, pseudoswarmers displayed increased activity, rather than transcription, of the flagellar master regulatory protein, FlhD 4 C 2 , and analyses of the fliL parent strain and its motile revertants showed that they result from mutations altering the C-terminal 14 amino acids of FliL. Collectively, the data suggest a functional role for the C terminus of FliL in surface sensing and implicate UmoA as part of the signal relay leading to the master flagellar regulator FlhD 4 C 2 , which ultimately controls swarmer cell differentiation. Proteus mirabilis is an enterobacterial opportunistic urinary tract pathogen that causes infections often associated with indwelling catheters or structural abnormalities of the urinary tract (reviewed in references 19 and 39). These infections are aided by several virulence factors, with one of the most significant being the ability of these bacteria to respond to the surfaces of host cells in a behavior referred to as swarming that allows P. mirabilis to move from an initial site of colonization (for example, a catheter surface) to uroepithelial cells of the urinary tract (3,30,41).Swarming is a flagellum-dependent motile behavior that is distinct from swimming in that it is a multicellular process that occurs on solid surfaces or in viscous liquids. Swarming behavior requires differentiation of vegetative swimmer cells into highly elongated, hyperflagellated swarmer cells (4, 31). Swarmer cell differentiation is initiated upon contact with a solid surface, which inhibits flagellar rotation, and an obligate requirement exists for this stimulus, as swarmer cells removed from a surface quickly dedifferentiated to vegetative swimmer cells (1, 23, 47, 52). Other conditions that inhibit flagellar rotation, such as addition of compounds that increase fluid visco...

Proteus mirabilis is a urinary tract pathogen and well known for its ability to move over agar surfaces by flagellum-dependent swarming motility. When P. mirabilis encounters a highly viscous environment, e.g., an agar surface, it differentiates from short rods with few flagella to elongated, highly flagellated cells that lack septa and contain multiple nucleoids. Proteus mirabilis is a Gram-negative enteric gammaproteobacterium that is well known for its ability to move over nutrient agars by flagellum-dependent swarming motility and for its role as an opportunistic pathogen in urinary tract infections (UTIs) (1, 2). P. mirabilis is dimorphic and exhibits two cell morphotypes whose expression is dependent on the surrounding environment. In liquid environments, i.e., nutrient broths, P. mirabilis exists as a short vegetative swimmer cell that is 1.5 to 2.0 m in length and possesses between 4 and 10 flagella per cell. When a swimmer cell encounters a viscous environment or a surface, e.g., nutrient agars, the swimmer cell differentiates into a swarmer cell 10 to 80 m in length, with multiple evenly spaced nucleoids within its cytoplasm, that is propelled by hundreds or thousands of flagella per cell (1,3,4). Swarmer cells are uniquely adapted to life on surfaces (3,(5)(6)(7)(8), and their ability to sense and respond to the host cell surface environment is thought to be critical in the regulation of several important virulence factors (1, 9-11).Expression of the genes associated with swarmer cell differentiation is induced when a swimmer cell contacts a surface, and this effect is termed surface sensing (1, 4, 5). More than 50 genes, including flagellar and virulence genes, are involved in P. mirabilis swarmer cell differentiation (4, 12, 13). Expression of the swarmer cell regulon is controlled in large measure by FlhD 4 C 2 , which is well-known as the master regulator of flagellar biosynthesis, acting to control a three-tiered hierarchical cascade of transcription (14-18). In this hierarchy, the flhDC operon (the sole member of class I) encodes the flagellar master regulator that in turn controls the expression of class II genes. Class II gene products include proteins for the assembly of the flagellar hook-basal body (HBB) complex, the type III protein export apparatus, and the regulators FliA and FlgM, which in turn control expression of class III genes (19,20). Class III genes encode proteins required for later steps in flagellar assembly, including motor and chemotaxis proteins, flagellar filament-associated proteins, and flagellin (FlaA), the major protein of the flagellum (18,20,21). When P. mirabilis differentiates from a swimmer to a swarmer cell, transcription at all levels of the flagellar regulon is upregulated to produce the hundreds of newly synthesized flagella on the swarmer cell (12, 13, 21). Inhibition of cell division and septation is also necessary to form polyploid swarmer cells and is controlled (in part) by FlhD 4 C 2 (22).Of the class II proteins, the function of FliL remains obscure. f...

Proteus mirabilis is a dimorphic motile bacterium well known for its flagellum-dependent swarming motility over surfaces. In liquid, P. mirabilis cells are 1.5-to 2.0-m swimmer cells with 4 to 6 flagella. When P. mirabilis encounters a solid surface, where flagellar rotation is limited, swimmer cells differentiate into elongated (10-to 80-m), highly flagellated swarmer cells. In order for P. mirabilis to swarm, it first needs to detect a surface. The ubiquitous but functionally enigmatic flagellar basal body protein FliL is involved in P. mirabilis surface sensing. Previous studies have suggested that FliL is essential for swarming through its involvement in viscosity-dependent monitoring of flagellar rotation. In this study, we constructed and characterized ⌬fliL mutants of P. mirabilis and Escherichia coli. Unexpectedly and unlike other fliL mutants, both P. mirabilis and E. coli ⌬fliL cells swarm (Swr ؉ ). Further analysis revealed that P. mirabilis ⌬fliL cells also exhibit an alteration in their ability to sense a surface: e.g., ⌬fliL P. mirabilis cells swarm precociously over surfaces with low viscosity that normally impede wild-type swarming. Precocious swarming is due to an increase in the number of elongated swarmer cells in the population. Loss of fliL also results in an inhibition of swarming at <30°C. E. coli ⌬fliL cells also exhibit temperature-sensitive swarming. These results suggest an involvement of FliL in the energetics and function of the flagellar motor. Most bacteria are able to live a planktonic free-living lifestyle or in a surface-attached microbial community called a "biofilm." The interchange between the motile and the sessile phases, referred to as the "swim-or-stick" switch, is not merely stochastic. Rather, the lifestyle change occurs in response to cues that a cell senses as it nears a surface (1). These surface signals are required and initiate biofilm formation (1). A fundamental question underlying the transition in lifestyle from motile to sessile phases is how does a bacterium sense a surface?Studies of many different bacterial species support the idea that surface sensing often involves the bacterial flagellum (2), which also facilitates movement toward and attachment to a surface. However, it is generally agreed that motility and biofilm formation are mutually exclusive. Moreover, flagella are used not only for swimming in liquid but also for swarming over a solid surface. Many bacterial species swarm, and often, as in Proteus mirabilis, require a surface-induced differentiated cell type, called a swarmer cell, that is elongated and hyperflagellated (3).P. mirabilis is a Gram-negative gammaproteobacterium belonging to the Enterobacteriaceae family. It is an opportunistic pathogen capable of causing urinary tract infections (UTI) (4-6). P. mirabilis is dimorphic and produces short vegetative swimmer cells (1.5 to 2.0 m in length) with a single nucleoid and 4 to 10 peritrichous flagella when cultured in nutrient broth. Conversely, when cultured on nutrient agar or in viscous en...

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